Early Detection of Salmonella spp. Contamination in
Raw Beef Meat Samples
Lizaïg Gouguet, Christelle Nahuet, Sébastien Bouton, Sirine Assaf and Sylvie Hallier-Soulier
Pall GeneDisc Technologies, Centre CICEA, 1 rue du Courtil, 35170 BRUZ, France. Contact email: genedisc@pall.com
Introduction
Results
Salmonella is a foodborne pathogen commonly found in the gastrointestinal
tract of healthy feedlot cattle and can be transferred to the carcass surface
during hide removal and evisceration procedures. Some current legislations
require industrials to guarantee absence of Salmonella spp. in 25 g sample
before product release.
Cultural methods, based on two successive enrichment steps (MLG 4, ISO 6578),
provide results in 3 and 5 days, for negative and positive samples, respectively.
Many alternative methods based on real-time Polymerase Chain Reaction (qPCR), certified by AFNOR Certification and/or AOAC-RI, have been developed.
For raw beef meat samples, they typically rely on a short enrichment step (10 ±
2 h) and enable results in 1 to 2 days for negative and positive samples,
respectively.
In order to allow meat industries to rapidly assess contamination level, a new
protocol was developed with a reduced time to result in as short as 4 h using
the GeneDisc® PCR based technology from Pall Corporation.
PCR Screening
Spiking Dose
Enrichment
Time
1 CFU/g
2h
100 CFU / 375 g
3h
10 CFU / 375 g
3h
5 CFU / 375 g
4h
1 - 2 CFU / 375 g
5h
Nbr. Presence/Total Nbr.
SAMPLES
stx+
Presence %
12 / 12
100 %
12 / 12
100 %
12 /15
80 %
23 / 24
95.8 %
15 / 15
100 %
94.9 %
of presence
of the PCR result by culture required a sub-culture of the
enrichment in RVS for 24 h at 41.5 °C
Confirmation
Main Effect Plot
Workflow
❶ Enrichment
❷ DNA Extraction (Extraction Pack Food 1)
• Bacterial concentration from 5 mL of enriched sample
» Centrifuge 5 min at 500 g
» Transfer of the supernatant into a sterile 15 mL tube
» Centrifuge 5 min at 10,000 g to pellet bacteria
» Suspension of the bacterial pellet with 200 µL of dilution
buffer
» Transfer of the bacterial suspension into a pre-dried lysis
tube
• Bacterial lysis
» Sonication for 5 min
» Heating at 102 oC for 10 min
❸ Real-Time PCR analysis (GeneDisc Cycler)
❹ Confirmation
• GeneDisc Plate Salmonella spp.
36.8
Ct*
Dilution: 25 -375 g raw beef meat + 225 mL - 1.5 L
BPW pre-warmed at 41.5 °C
Incubation: 2 - 5 h at 41.5 o C ± 1 o C
*Ct (cycle threshold) : The number of cycles required for the fluorescent signal to exceeds background level.
No
No
impact of the matrix
impact of the Salmonella strain
Mean Ct values about 36.8 for all the enrichment times/spiking doses
Enrichment Time and Level of Salmonella spp.
Contamination
1H
2H
3H
4H
5H
ENRICHMENT TIME
1 CFU/g
• Secondary enrichment into Rappaport-Vassiliadis Soya
broth (10 mL)
and/or
• Plating on chromogenic media (XLD)
100 CFU/sample
10 CFU/sample
5 CFU/sample
1-2 CFU/sample
Spiking Conditions
Example of positive PCR signal
Bolognese ground Beef 375 g
1 CFU S. enterica Arizonae / 375 g
5 h enrichment time
Spiking conditions
• Spiking doses: 1-2, 5, 10, 100 CFU/samples and 1 CFU/g
• Injury level of the Salmonella cells: 33 – 79 %
Conclusion
Salmonella spp. strains:
• Salmonella enterica Typhimurium
• Salmonella enterica Houtanae
• Salmonella enterica Arizonae
Samples (375 g / 5-20 % fat):
• Raw ground beef from butcher and
supermarket
• Raw beef trim from butcher and
supermarket
• Seasoned raw ground beef from
supermarket
• Frozen ground beef (supermarket)
Results obtained with 78 different samples highlighted that the sample type
(fresh ground beef, seasoned raw ground beef, beef trim , frozen ground beef)
and/or the Salmonella spp. injury level had no impact on the enrichment time
needed for detection. Thus, the enrichment time for the detection of Salmonella
spp. in raw beef samples only depended on the initial Salmonella spp.
contamination level.
With this new protocol for the GeneDisc method, beef meat processors can
now increase their profitability by implementing tests that fit each process stage
in terms of sensitivity and rapidity.
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